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* Flow cytometry is a powerful tool for interrogating the phenotype and characteristics of cells. It is based upon the light-scattering properties of the cells being analyzed and these include fluorescence emissions. This fluorescence may be associated with dyes or conjugated to mAbs specific for molecules either on the surface or in the intracellular components of the cell 14

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* Flow cytometry facilitates the identification of different cell types within a heterogeneous population * It was initially developed by immunologists wishing to separate out different cell populations for subsequent coculture experiments to determine the function of cells within the immune system * This was achieved by using fluorescence activated cell sorting, or FACS, on the flow cytometer. * The initial instruments were able to analyze one or two colors of fluorescence; today, instruments capable of analyzing 11 colors of fluorescence are available 15

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Flow Cytometer Instrumentation Graphical Summary 17

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* All forms of cytometry depend on the basic laws of physics, including those of fluidics, optics, and electronics * Flow cytometry is a system for sensing cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area 18

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* The amount of light scattered to the side is detected in the side or 90 o scatter channel * influenced by the granularity of cells * used to distinguish granulated cells from non-granulated cells 26

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1. Cells in suspension flow single file past 2. Focused laser where they scatter light and emit fluorescence that is filtered and collected 3. Then converted to digitized values that are stored in a file Optic Fluidics Electronics Flow Cytometers are made of 31

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* Need to have cells in suspension flow in single file * The cells from the sample tube are injected into the sheath fluid (PBS) * Flow in a flow cell is laminar. * Laminar flow: sample fluid flows in a central core that does not mix with the sheath fluid 32

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* The flow chamber is instrumental in delivering the cells in suspension to the specific point that is intersected by the illuminating beam and the plane of focus of the optical assembly * Cells suspended in isotonic fluid are transported through the sensing system * To confine cells to the center of the flow stream; this also reduces blockage due to clumping 35

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Sheath Sample Stream Cell The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing. 36

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* Fluorochromes on/in the cell may absorb some of the light and become excited * As those fluorochromes leave their excited state, they release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength 39

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Flow Cell Injector Tip Focused laser beam Sheath fluid 40

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* Many wavelengths of light will be scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters * Optical filters : absorb or reflect some wavelengths of light, while transmitting other. 41

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For each color, adjust voltage so that the negative population is in the first decade, off of the axis (if possible) 2- Setting Starting Voltages for Fluorescent Parameters 60

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* Negative” signal on cells is auto fluorescence due to flavins, porphyrins and other molecules or properties of the material (plastics fluoresce in certain excitation wavelengths). * Different cells will have different levels of autofluorescence (e.g.lymphs vs. monos, different cell lines) affecting sensitivity in certain parameters with high base signals. 61

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 Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more)  Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome  You need to “compensate” for this bleed over so that 1 detector reports signal from only 1 fluorochrome 62

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 There is some overlap between the colors emitted by different fluorescent markers, therefore mathematical compensation is used to reduce overlapping results 63

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BD LSR II User’s Guide; Becton Dickinson 66

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* The threshold can be set on any parameter, but is usually set on FSC 67